Tmem174, a regulator of phosphate transporter prevents hyperphosphatemia

Renal type II sodium-dependent inorganic phosphate (Pi) transporters NaPi2a and NaPi2c cooperate with other organs to strictly regulate the plasma Pi concentration. A high Pi load induces expression and secretion of the phosphaturic hormones parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) that enhance urinary Pi excretion and prevent the onset of hyperphosphatemia. How FGF23 secretion from bone is increased by a high Pi load and the setpoint of the plasma Pi concentration, however, are unclear. Here, we investigated the role of Transmembrane protein 174 (Tmem174) and observed evidence for gene co-expression networks in NaPi2a and NaPi2c function. Tmem174 is localized in the renal proximal tubules and interacts with NaPi2a, but not NaPi2c. In Tmem174-knockout (KO) mice, the serum FGF23 concentration was markedly increased but increased Pi excretion and hypophosphatemia were not observed. In addition, Tmem174-KO mice exhibit reduced NaPi2a responsiveness to FGF23 and PTH administration. Furthermore, a dietary Pi load causes marked hyperphosphatemia and abnormal NaPi2a regulation in Tmem174-KO mice. Thus, Tmem174 is thought to be associated with FGF23 induction in bones and the regulation of NaPi2a to prevent an increase in the plasma Pi concentration due to a high Pi load and kidney injury.


Results
COXPRESdb search indicates co-expression of transmembrane protein 174 (Tmem174) with renal NaPi transporters. To identify genes co-regulated with slc34a1 or slc34a3 mouse NaPi transporters, we searched for genes using the COXPRESdb v7 15 . The top 20 genes are listed in Supplemental Tables S1 and S2, and the transmembrane protein 174 (Tmem174) gene was identified as a significant gene co-expressed with slc34a1 and slc34A3. The correlation coefficient (r) for gene expression levels between slc34a1 or slc34a3 and Tmem174 was 0.90 and 0.37, respectively (Fig. 1a,b).
TMEM174 was originally identified among a large pool of genes by high-throughput cell screening technology to isolate functional genes and provide insight into the mechanisms of gene function [17][18][19] . The full-length amino acid sequences of Tmem174 in mouse (NP_080961.1), rat (NP_001019469.1), and human (NP_694949.1) are reported in the NCBI database. The putative Tmem174 protein comprises 243 amino acids with 2 transmembrane domains.
Tissue localization of Tmem174 expression and possible involvement in Pi homeostasis. Expression of Tmem174 mRNA was analyzed by real-time polymerase chain reaction (PCR) using mouse tissues. As reported in human tissue 17 , mouse Tmem174 mRNA was markedly higher in the kidney compared with other tissues (Fig. 2a). Tmem174 protein expression was detected at the apical membrane of renal proximal tubular cells, but not in the distal tubule (Fig. 2b). Next, we examined whether the renal Tmem174 protein expression was regulated by dietary Pi regulation and deletion of renal Pi transporters NaPi2a, or NaPi2c ( Fig. 2c-e). A low Pi (LP) diet significantly induced renal Tmem174 protein expression compared with control Pi (CP) and high Pi (HP) diets, similar to the response of renal Pi transporters to dietary Pi content (Fig. 2c,d). Furthermore, deletion of renal NaPi transporters (NaPi2a-KO and NaPi2c-KO mice) significantly reduced the renal Tmem174 protein expression levels compared with NaPi2a +/+ NaPi2c +/+ mice (Fig. 2e).
Characterization of Tmem174 −/− mice fed standard mouse chow. To generate Tmem174-null mice, we replaced the genomic region extending from Tmem174 exon 1 to the 5' portion of exon 2 with a neomycinresistant gene (Supplemental Fig. S1a). We confirmed the mutant genomic DNA isolated from transfected ES clones by Southern blot analysis, and the mice genotype by PCR analysis (Supplemental Fig. S1b,c). Reverse transcriptase-PCR with Tmem174-specific primers and Western blotting analysis confirmed the absence of detectable renal Tmem174 mRNA and protein expression in Tmem174 −/− mice (Fig. 3a,b). Male and female Tmem174 −/− mice showed similar weight gain compared with Tmem174 +/+ and Tmem174 +/mice (Fig. 3c). To measure food intake, and urine and fecal biochemical data, mice were individually placed in metabolic cages. Tmem174 −/− mice did not show a significant difference in food intake, plasma creatinine, plasma blood urea a b r=0.90 www.nature.com/scientificreports/ nitrogen (BUN), blood ionized Ca and plasma Pi concentrations, fecal and urinary Ca, Pi excretion levels, or other blood biochemistry parameters compared with Tmem174 +/+ and Tmem174 −/− mice ( Fig. 3d-l, and Supplemental Table S3).
Trends in Pi-regulating hormones in Tmem174 −/− mice. Plasma 1,25(OH) 2 D levels were not significantly different among the 3 groups (Fig. 4a). Plasma PTH, and especially serum intact FGF23 levels were markedly higher in Tmem174 −/− mice than in Tmem174 +/+ and Tmem174 +/mice (Fig. 4b,c). Renal 25-hydroxyvitamin D-1 alpha hydroxylase (Cyp27b1) mRNA levels were not significantly different among the 3 groups, but renal 25-hydroxyvitamin D-24-hydroxylase (Cyp24a1) mRNA levels were significantly higher in Tmem174 −/− mice than in Tmem174 +/+ and Tmem174 +/mice (Fig. 4d,e). Parathyroid PTH mRNA levels were not significantly different among the three groups (Fig. 4f). FGF23 mRNA was mainly detected in osseous-tissues but has been found in other tissues as well 20 . Expression of FGF23 mRNA in the bone was highly increased in Tmem174 −/− mice compared with Tmem174 +/+ mice, and to a lower extent in the spleen and thymus (Fig. 4g). FGF23 mRNA levels in the kidney of Tmem174 −/− mice tended to be increased compared with that in control mice but were very low compared with that in the bone tissue. In both Tmem174 +/+ and Tmem174 −/− mice, FGF23 immunostaining was observed in osteocytes and osteoblasts/preosteoblasts, with no difference in the localization patterns. The number of FGF23-positive cells, however, tended to be increased in Tmem174 −/− mice (Fig. 4h).    Fig. S2). Hematoxylin and eosin staining of the femurs from 8-weekold mice revealed that Tmem174 −/− mice had slightly more cancellous bone at the metaphysis compared with Tmem174 +/+ mice (Fig. 5a). Consistently, bone histomorphometry showed that the BV/TV was greater in 8-week-old Tmem174 −/− mice than in Tmem174 +/+ mice (Supplemental Fig. S2). There are no significant differences in Tb. Th, Ct. Th., or the width of growth plate between the Tmem174 +/+ and Tmem174 −/− mice. In addition, alkaline phosphatase (ALP)/tartrate-resistant acid phosphatase staining revealed a tendency toward a thicker ALP-positive osteoblast/preosteoblast layer on the trabecular surface in Tmem174 −/− mice, and a similar number of tartrate-resistant acid phosphatase-positive osteoclasts between Tmem174 +/+ and Tmem174 −/− mice, or a slightly higher number than in Tmem174 −/− mice (Fig. 5b). Furthermore, the bone mineralization and osteoid layer thickness did not differ significantly between Tmem174 +/+ and Tmem174 −/− mice, whereas mature osteoblasts covered the bone surface in Tmem174 −/− mice (Fig. 5c).
Next, we examined the interaction between Tmem174 and NaPi2a/NHERF1 using renal BBMVs of wildtype (WT), Tmem174-KO, and NaPi2a-KO mice (Fig. 7c,b). NHERF1 immunoprecipitation analysis revealed a NaPi2a/NHERF1 interaction in both WT and Tmem174-KO mice. In contrast, NHERF1 immunoprecipitation analysis detected Tmem174 protein in WT mice, but not in NaPi2a-KO mice (Fig. 7c). These findings suggest that www.nature.com/scientificreports/ Tmem174 binds to NHERF1 in the presence of NaPi2a but cannot interact in the absence of NaPi2a. Tmem174 immunoprecipitation analyses showed an interaction between Tmem174 and NaPi2a (Fig. 7d).

Response to the dietary Pi content in Tmem174 KO mice. We examined fluctuations in the plasma
Pi levels due to differences in the dietary Pi content (Fig. 8a). Tmem174 −/− mice fed the HP diet had markedly higher plasma Pi levels than Tmem174 +/+ mice. Furthermore, Tmem174 −/− mice fed the CP or HP diets had extremely high serum FGF23 levels compared with Tmem174 +/+ mice fed an equivalent diet (Fig. 8b). In addition, renal NaPi2a, but not NaPi2c, protein expression levels were significantly higher in Tmem174 −/− mice fed  www.nature.com/scientificreports/ the LP, CP, or HP diet compared with Tmem174 +/+ mice fed the same diet (Fig. 8c). In contrast, the CP diet and HP diet significantly suppressed renal NaPi2c protein expression levels in Tmem174 −/− mice compared with Tmem174 +/+ mice (Fig. 8c). NaPi2a immunofluorescence staining was strongly detected at the apical membrane of proximal tubular cells both in Tmem174 +/+ and Tmem174 −/− mice fed the LP diet (Fig. 8d). The HP diet suppressed NaPi2a staining at the apical membrane in Tmem174 +/+ mice. In contrast, Tmem174 −/− mice fed the HP diet maintained NaPi2a strong immunostaining at the apical membrane of the proximal tubular cells (Fig. 8d).
Response to FGF23 in Tmem174 KO mice. Next, to confirm the effect of the phosphaturic action of FGF23 in Tmem174 −/− mice, mice were fed the LP diet to reduce endogenous FGF23. Exogenous FGF23 was expressed using the Naked-DNA method, as described previously [24][25][26] . As shown in Supplemental Fig. S4a, the LP diet significantly suppressed serum FGF23 levels in both Tmem174 +/+ and Tmem174 −/− mice. We confirmed the exogenous FGF23 mRNA (hFGF23) expression in the liver of the FGF23 groups in both Tmem174 +/+ and Tmem174 −/− mice at 4 days after Naked-DNA injection (Supplemental Fig. S4b) [24][25][26] . In Tmem174 +/+ mice, but not in Tmem174 −/− mice, FGF23 increased the level of ERK1/2 phosphorylation compared with the control group (Fig. 9a). Interestingly, the ERK phosphorylation level was higher in the control Tmem174 −/− mice compared with the control Tmem174 +/+ mice. FGF23 groups of both Tmem174 +/+ and Tmem174 −/− mice showed significantly lower levels of renal α-Klotho protein expression compared with their control groups (Supplemental Fig. S4c). Renal Cyp27b1 mRNA levels were significantly suppressed, and Cyp24a1 mRNA levels were significantly increased in the FGF23 groups of both Tmem174 +/+ and Tmem174 −/− mice compared with their control groups (Supplemental Fig. S4d,e). Slc34a1, but not slc34a3, mRNA levels were significantly suppressed in FGF23 groups of both Tmem174 +/+ and Tmem174 −/− mice compared with the control group (Supplemental Fig. S4f,g). Urinary Pi excretion levels were slightly but significantly increased in FGF23 groups of both Tmem174 +/+ and Tmem174 −/− mice compared with the control group (Fig. 9b). In Tmem174 +/+ mice, both renal NaPi2a and NaPi2c protein levels were significantly suppressed after FGF23 Naked DNA injection compared with the control group (Fig. 9c). In Tmem174 −/− mice, FGF23 significantly suppressed only NaPi2c protein expression and not NaPi2a protein expression levels (Fig. 9c).

Renal injury in Tmem174 KO mice. Finally, we investigated the plasma Pi and BUN concentrations in
a folic acid (FA)-induced acute kidney injury (AKI) model for 7 days. As shown in Fig. 10a, the deletion of Tmem174 shortened the lifespan of the AKI model mice. There were no significant differences in BUN levels at 24 h and 7 days after administration of FA between Tmem174 +/+ and Tmem174 −/− mice (Fig. 10b,c). AKI-Tmem174 −/− mice, however, had significantly higher levels of plasma Pi at 24 h after FA administration compared with AKI-Tmem174 +/+ mice, and the hyperphosphatemia was maintained only in Tmem174 −/− mice until 7 days after FA treatment (Fig. 10d,e). Furthermore, serum intact FGF23 levels were markedly higher in Tmem174 −/− mice at 24 h after FA treatment compared with Tmem174 +/+ mice, and the markedly high levels of serum FGF23 were maintained only in Tmem174 −/− mice until 7 days after FA treatment (Fig. 10f,g). Renal NaPi2a protein levels were significantly reduced in Tmem174 +/+ mice by FA administration, but only slightly reduced in Tmem174 −/− mice (Fig. 10h).

Discussion
In the present study, we investigated the roles of a strongly correlated molecule (Tmem174) in the GCNs and Pi metabolism. Many transmembrane proteins grouped in the TMEM family are poorly described and have mostly unknown functions. After further characterization, they are generally renamed and reclassified into more specific categories such as GPCR proteins or ion channels 28,29 . The Tmem174 protein was extremely highly expressed in the kidney and localized at the apical membrane of the renal proximal tubules. Dietary Pi content regulates the renal Tmem174 protein levels the same as NaPi2a. Immunoprecipitation experiments suggest that Tmem174 interacts with the NaPi2a/NHERF1 complex.  (2) dietary Pi response abnormalities in the regulation of the plasma Pi concentration. The present findings suggest that Tmem174 binds to NaPi2a on the cell membrane and is involved in the internalization of NaPi2a. In Tmem174 −/− mice, no decrease in NaPi2a was observed despite high FGF23 and PTH concentrations. On the other hand, NaPi2c was significantly decreased. In addition, NHERF1 was significantly elevated in Tmem174 −/− mice. For these reasons, Tmem174 is considered to be a component of the NaPi2a/NHERF1 complex that receives signals from PTH and FGF23.
Although Tmem174 deficiency affects the NaPi2a/NHERF1 system and vitamin D-metabolizing enzymes, NaPi2c regulation are considered normal. PTH and FGF23 downregulate the NaPi2a/NHERF1 binary complex by activating 2 distinct signaling pathways that converge at NHERF1 1 . The internalization and degradation of NaPi2a increase Pi excretion and depend on activation of the ERK1/2 and serum/glucocorticoid-regulated kinase-1 pathways, resulting in phosphorylation of NHERF1 30 . PTH signals activate protein kinases A and C 12,31,32 . Triggered by phosphorylation of NHERF1, NaPi2a dissociates from NHERF1 and is then internalized 12 . Not all signals from the receptor have been examined in detail, but some signals were activated. In addition, Tmem174 −/− mice show an increase in renal cyp24a1 mRNA levels resulting from abnormally high serum FGF23 levels, but normal plasma 1,25(OH) 2 D levels. It is not clear why the plasma 1.25(OH) 2 D levels are not decreased. Certainly, suppression of cyp27b1 mRNA has not been observed, but because cyp24a1, which is involved in the degradation of 1,25 (OH) 2 D, is increased, it is assumed that plasma 1,25 (OH) 2 D levels would decrease. As expected, Tmem174 deficiency may affect the function of cyp24a1 (or cyp27b1) and the cellular metabolism of 1,25(OH) 2 D. As a member of the Tmem family, it is possible that it affects vitamin D-metabolizing enzymes as a constituent protein of intracellular organelles. Further studies on the role of Tmem174 in active vitamin D metabolism are needed.
In Tmem174 −/− mice, NaPi2c protein was significantly reduced as compared with NaPi2a protein. The interaction of NaPi2c with NHERF3 (PDZK1) is more important than that with NHERF1 33,34 . In fact, NaPi2c expression is suppressed in NHERF3-KO mice 34 . We previously reported differences in signals between the phosphaturic action of FGF23 and the inhibitory effect on vitamin D synthesis 35 . Therefore, it is considered that the effect of www.nature.com/scientificreports/ Tmem174 deficiency is limited to the control function of NaPi2a. More detailed studies on the role of Tmem174 in NaPi2a regulation, such as the effect of NHERF1 on phosphorylation, are needed. Another feature of Tmem174 −/− mice is enhanced FGF23 induction from the bone. High serum FGF23 levels cause the pathology observed in a mouse model of X-linked hypophosphatemia rickets (Hyp mice) 36,37 . On the other hand, Tmem174 −/− mice did not exhibit the abnormal bone morphology seen in Hyp mice and we speculate that this is because Tmem174 −/− mice do not develop hypophosphatemia. The bone analysis data suggest that a high PTH concentration affects fluctuations in the numbers of osteoblasts and osteoclasts. More detailed studies will help to clarify the role of Tmem174 in bone.
High FGF23 induction in Tmem174 −/− mice is improved by a low Pi diet. Therefore, renal Tmem174 is expected to signal dietary Pi levels to bone FGF23. On the other hand, in an FA-induced renal disorder model, a further increase in serum FGF23 concentration was observed in Tmem174 −/− mice. FGF23 induction is known to be independent of dietary Pi signals in an FA-acute kidney injury model 38 . Therefore, the increase in FGF23 in Tmem174 −/− mice may be independent of the signal of renal damage. The relationship between α-Klotho and Tmem174 as a mediator from the kidney remains unclear. α-Klotho plays an important role in phosphate regulation by FGF23 as a co-receptor for FGFR1 in the kidney 39,40 . In Tmem174 −/− mice, renal α-Klotho levels are reduced by approximately 50%. Previous studies reported that a decrease in α-Klotho in the kidney triggers the induction of FGF23 from the bone 39,40 . In contrast, we speculate that the cause of the α-Klotho decrease in the Tmem174 −/− mouse kidney is the high concentration of serum FGF23. There are many possible causes for the decrease in klotho, but it is unclear from this study.
Finally, phosphaturic hormone is secreted in response to an excessive Pi load and acts on the kidneys to promote Pi excretion. The NaPi2a/NHERF1 complex has an important role. Tmem174 is expected to regulate the amount of NaPi2a in response to a Pi deficiency or excess and regulates the responsiveness of phosphaturic hormone. For example, vitamin D treatment in Hyp mice restores serum Pi levels by causing FGF23 resistance to NaPi2a/NHERF1 35,41 . Thus, Tmem174, a strongly correlated molecule with NaPi2a in the GCNs, is thought to be involved in the regulation of NaPi2a by PTH and FGF23 in the kidney and the prevention of hyperphosphatemia in response to a high dietary Pi load (Fig. 11).

Materials and methods
Experimental animals. All experimental procedures involving animals were conducted in accordance with the Tokushima University School of Medicine and Osaka University Graduate School of Medicine guidelines. This study was also carried out in compliance with the ARRIVE guidelines. All procedures involving the use of animals were subjected to approval from Tokushima University School of Medicine (T2019-126) and Osaka University Graduate School of Medicine (19-064-02) ethics committee.
Male and female C57B6/J mice were purchased from Charles River Laboratories Japan (Yokohama, Japan). NaPi2a-KO and NaPi2c-KO mice were maintained as described previously 7,42 . Mice were weaned at 4 weeks of age and provided free access to water and standard mouse chow containing 0.8% phosphorus (Oriental MF; ORIENTAL YEAST CO., LTD, Osaka, Japan).
Generation of Tmem174-KO mice. Tmem174-deficient mice were generated by gene targeting. A targeting vector was constructed by replacing the 129 genomic Tmem174 loci (exon1 through part of exon2) with neo r (Supplemental Fig. S1a). The targeting vector was introduced into 129 days 14 embryonic stem (ES) cells by electroporation, and clones that underwent homologous recombination were confirmed by Southern blot analysis (Supplemental Fig. S1b). Genomic DNA was extracted from tail clippings and amplified by PCR using specific primers (Supplemental Fig. S1c, Table 1).

Effect of FGF23 and PTH on the regulation of NaPi transporter degradation. For exogenous
FGF23 expression, TransIT-EE Hydrodynamic Delivery Solution was used as the TransIT in vivo gene delivery system (TaKaRa Bio, Shiga, Japan) as described previously 25,26 . For PTH injection, mice were administered bovine PTH (amino acid 1-34; Sigma-Aldrich, St. Louis, MO, USA) at a dose of 7.5 μg/100 g body weight, as described previously 46,47 .
Metabolic cages to collect urine and fecal samples. Mice were individually placed in the metabolic cages at AM.10:00 for quantitative urine and fecal collection for 24 h with free access to food and water. Fecal samples were ashed according to a modified protocol, as described previously 35,43,48,49 . Biochemical measurements. Concentrations of Pi, Ca, and creatinine and BUN were determined using commercial kits (WAKO, Osaka, Japan). The fractional excretion index for Ca (FEICa) and Pi (FEIPi) was calculated as follows: urinary Ca or Pi/ (urinary creatinine /serum Ca or Pi). Concentrations of serum FGF23, plasma PTH, and 1,25(OH) 2 D were determined using the FGF23 ELISA kit (KAINOS Laboratories, Tokyo, Japan), intact PTH ELISA kit (Immunotopics Inc., San Clemente, CA, USA), and1,25-(OH) 2 Vitamin D ELISA Kit (Immundiagnostik, Bensheim, Germany), respectively. Other blood clinical parameters were analyzed by automated methods. The renal and urinary cAMP levels were measured using Cyclic AMP Select EIA Kit (Cayman Chemical, Ann Arbor, MI, USA). Kidneys were flash-frozen in liquid nitrogen and dropped into 5-10 volumes of 5% trichloroacetic acid (TCA) solution. The kidneys were homogenized on ice, and the supernatant was transferred to a clean tube after removing the precipitate by centrifugation at 1500×g for 10 min. TCA was extracted from the samples using water-saturated ether. The samples were further heated at 70 °C for 5 min to remove any residual ether in the aqueous layer. The supernatant of the kidney extract was used for cAMP measurement according to the manufacturer's instructions.
RNA extraction, cDNA synthesis, and quantitative PCR. Mouse tissues were sampled, immediately submerged in RNAlater (Sigma-Aldrich), and stored at − 20 °C until use. Total cellular RNA from the sampled tissues was extracted and purified using ISOGEN (Wako, Osaka Japan) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized as described previously 35,43,48,49 . The template DNA was omitted for the negative control (-) for all PCR experiments. The PCR reaction was examined without reverse transcriptase (data not shown). Quantitative PCR was performed using ABI PRISM 7500 (Applied Biosystems, Foster City, CA) as described previously 35,43,45,48,49 . The reaction mixture consisted of 10 μl of SYBR Premix Ex Taq, ROX Reference Dye II (Perfect Real Time, TaKaRa Bio) with specific primers (Table 2).
Protein sample purification and immunoblotting. BBMVs prepared using the Ca 2+ precipitation method, cortical membrane, and whole homogenate were obtained from mouse kidneys and used for immunoblotting and immunoprecipitation analyses as described previously 35,45,48,49 . Protein samples were heated at